Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Protein interaction partner”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 469 records · Page 26Linked to original sources
PubMed2002

Spectral profiling for the simultaneous observation of four distinct fluorescent proteins and detection of protein-protein interaction via fluorescence resonance energy transfer in tobacco leaf nuclei.

The control of subcellular localization of proteins and their interaction with other partners in vivo are important parameters that provide clues to their function and regulation. The ability to simultaneously track multiple protein species with high resolution should provide a valuable assay system to study and characterize various types of posttranslational control pathways. In this work, we established the system and a method involving "spectral profiling" for the resolution of four different fluorescent protein tags in the same viewing field using digital imaging technology. With these techniques, we have (a) developed new derivatives of mGFP5, which is commonly used in the plant field, that are about three times brighter; (b) demonstrated that four spectrally distinct fluorescent proteins (cyan, green, yellow, and red) that are fused to a transcription factor could be stably expressed in nuclei and distinguished in tobacco (Nicotiana tabacum) mesophyll cells; and (c) shown that interaction between partners of a dimeric transcription factor can be detected by measuring fluorescence resonance energy transfer. These technologies should help one to study protein-protein interactions efficiently, especially for nuclear proteins under in vivo conditions.

Cell Nucleus↗
PubMed2005

Physicochemical and residue conservation calculations to improve the ranking of protein-protein docking solutions.

Many protein-protein docking algorithms generate numerous possible complex structures with only a few of them resembling the native structure. The major challenge is choosing the near-native structures from the generated set. Recently it has been observed that the density of conserved residue positions is higher at the interface regions of interacting protein surfaces, except for antibody-antigen complexes, where a very low number of conserved positions is observed at the interface regions. In the present study we have used this observation to identify putative interacting regions on the surface of interacting partners. We studied 59 protein complexes, used previously as a benchmark data set for docking investigations. We computed conservation indices of residue positions on the surfaces of interacting proteins using available homologous sequences and used this information to filter out from 56% to 86% of generated docked models, retaining near-native structures for further evaluation. We used a reverse filter of conservation score to filter out the majority of nonnative antigen-antibody complex structures. For each docked model in the filtered subsets, we relaxed the conformation of the side chains by minimizing the energy with CHARMM, and then calculated the binding free energy using a generalized Born method and solvent-accessible surface area calculations. Using the free energy along with conservation information and other descriptors used in the literature for ranking docking solutions, such as shape complementarity and pair potentials, we developed a global ranking procedure that significantly improves the docking results by giving top ranks to near-native complex structures.

Algorithms↗
PubMed2004

Extensive domain motion and electron transfer in the human electron transferring flavoprotein.medium chain Acyl-CoA dehydrogenase complex.

The crystal structure of the human electron transferring flavoprotein (ETF).medium chain acyl-CoA dehydrogenase (MCAD) complex reveals a dual mode of protein-protein interaction, imparting both specificity and promiscuity in the interaction of ETF with a range of structurally distinct primary dehydrogenases. ETF partitions the functions of partner binding and electron transfer between (i) the recognition loop, which acts as a static anchor at the ETF.MCAD interface, and (ii) the highly mobile redox active FAD domain. Together, these enable the FAD domain of ETF to sample a range of conformations, some compatible with fast interprotein electron transfer. Disorders in amino acid or fatty acid catabolism can be attributed to mutations at the protein-protein interface. Crucially, complex formation triggers mobility of the FAD domain, an induced disorder that contrasts with general models of protein-protein interaction by induced fit mechanisms. The subsequent interfacial motion in the MCAD.ETF complex is the basis for the interaction of ETF with structurally diverse protein partners. Solution studies using ETF and MCAD with mutations at the protein-protein interface support this dynamic model and indicate ionic interactions between MCAD Glu(212) and ETF Arg alpha(249) are likely to transiently stabilize productive conformations of the FAD domain leading to enhanced electron transfer rates between both partners.

Acyl-CoA Dehydrogenase↗
PubMed2003

Peptide libraries: at the crossroads of proteomics and bioinformatics.

Peptide libraries offer a valuable means for providing functional information regarding protein-modifying enzymes and protein interaction domains. Library approaches have become increasingly useful as high-throughput strategies for the analysis of large numbers of new proteins identified as a result of genome-sequencing efforts. Recent developments in the field have produced faster methods with broadened applicability. Crucially, new computational and biochemical tools have emerged that facilitate identification of interaction partners and substrates for proteins on the basis of their peptide selectivity profiles. Such combinations of proteomics-scale experimental approaches with bioinformatics tools hold great promise for the elucidation of protein interaction networks and signal transduction pathways in living cells.

Computational Biology↗
PubMed2002

Identification of Ca2+-dependent binding partners for the neuronal calcium sensor protein neurocalcin delta: interaction with actin, clathrin and tubulin.

The neuronal calcium sensors are a family of EF-hand-containing Ca(2+)-binding proteins expressed predominantly in retinal photoreceptors and neurons. One of the family members is neurocalcin delta, the function of which is unknown. As an approach to elucidating the protein interactions made by neurocalcin delta, we have identified brain cytosolic proteins that bind to neurocalcin delta in a Ca(2+)-dependent manner. We used immobilized recombinant myristoylated neurocalcin delta combined with protein identification using MS. We demonstrate a specific interaction with clathrin heavy chain, alpha- and beta-tubulin, and actin. These interactions were dependent upon myristoylation of neurocalcin delta indicating that the N-terminal myristoyl group may be important for protein-protein interactions in addition to membrane association. Direct binding of neurocalcin delta to clathrin, tubulin and actin was confirmed using an overlay assay. These interactions were also demonstrated for endogenous neurocalcin delta by co-immunoprecipitation from rat brain cytosol. When expressed in HeLa cells, neurocalcin delta was cytosolic at resting Ca(2+) levels but translocated to membranes, including a perinuclear compartment (trans-Golgi network) where it co-localized with clathrin, following Ca(2+) elevation. These data suggest the possibility that neurocalcin delta functions in the control of clathrin-coated vesicle traffic.

Actins↗
PubMed2005

Distinct protein classes including novel merozoite surface antigens in Raft-like membranes of Plasmodium falciparum.

Glycosylphosphatidylinositol (GPI)-anchored proteins coat the surface of extracellular Plasmodium falciparum merozoites, of which several are highly validated candidates for inclusion in a blood-stage malaria vaccine. Here we determined the proteome of gradient-purified detergent-resistant membranes of mature blood-stage parasites and found that these membranes are greatly enriched in GPI-anchored proteins and their putative interacting partners. Also prominent in detergent-resistant membranes are apical organelle (rhoptry), multimembrane-spanning, and proteins destined for export into the host erythrocyte cytosol. Four new GPI-anchored proteins were identified, and a number of other novel proteins that are predicted to localize to the merozoite surface and/or apical organelles were detected. Three of the putative surface proteins possessed six-cysteine (Cys6) motifs, a distinct fold found in adhesive surface proteins expressed in other life stages. All three Cys6 proteins, termed Pf12, Pf38, and Pf41, were validated as merozoite surface antigens recognized strongly by antibodies present in naturally infected individuals. In addition to the merozoite surface, Pf38 was particularly prominent in the secretory apical organelles. A different cysteine-rich putative GPI-anchored protein, Pf92, was also localized to the merozoite surface. This insight into merozoite surfaces provides new opportunities for understanding both erythrocyte invasion and anti-parasite immunity.

Amino Acid Motifs↗
PubMed2005

Interaction of Huntingtin-associated protein-1 with kinesin light chain: implications in intracellular trafficking in neurons.

Huntingtin-associated protein-1 (HAP1) was initially identified as an interacting partner of huntingtin, the Huntington disease protein. Unlike huntingtin that is ubiquitously expressed throughout the brain and body, HAP1 is enriched in neurons, suggesting that its dysfunction could contribute to Huntington disease neuropathology. Growing evidence has demonstrated that HAP1 and huntingtin are anterogradely transported in axons and that the abnormal interaction between mutant huntingtin and HAP1 may impair axonal transport. However, the exact role of HAP1 in anterograde transport remains unclear. Here we report that HAP1 interacts with kinesin light chain, a subunit of the kinesin motor complex that drives anterograde transport along microtubules in neuronal processes. The interaction of HAP1 with kinesin light chain is demonstrated via a yeast two-hybrid assay, glutathione S-transferase pull down, and coimmunoprecipitation. Furthermore, HAP1 is colocalized with kinesin in growth cones of neuronal cells. We also demonstrated that knocking down HAP1 via small interfering RNA suppresses neurite outgrowth of PC12 cells. Analysis of live neuronal cells with fluorescence microscopy and fluorescence recovery after photobleaching demonstrates that suppressing the expression of HAP1 or deleting the HAP1 gene inhibits the kinesin-dependent transport of amyloid precursor protein vesicles. These studies provide a molecular basis for the participation of HAP1 in anterograde transport in neuronal cells.

Amyloid beta-Protein Precursor↗
PubMed2004

Oligomerization activity of a double-stranded RNA-binding domain.

Xenopus laevis RNA-binding protein A (Xlrbpa) is a highly conserved, ubiquitously expressed hnRNP- and ribosome-associated RNA-binding protein that contains three double stranded RNA-binding domains (dsRBDs) in tandem arrangement. A two-hybrid screen with Xlrbpa as a bait recovered Xlrbpa itself as the strongest interaction partner, indicating multimerization of this protein. To search for regions responsible for the observed interaction, we conducted two-hybrid assays with Xlrbpa deletion constructs and identified the third dsRBD of Xlrbpa as the exclusive interacting domain. Additionally, these results were confirmed by coimmunoprecipitation experiments with truncated proteins expressed both in yeast and Xenopus oocytes. In PACT, the human homologue of Xlrbpa, we could demonstrate that the third dsRBD displays the same multimerization activity. Interestingly, this domain is essential for the activation of the dsRNA-activated protein kinase PKR. Addition of RNAses to coimmunoprecipitation experiments did not affect the dimerization, suggesting that the interaction is independent of RNA-binding. We report here a homomultimerization activity of a type B dsRBD and suggest possible implications that include a model for PKR activation by PACT.

Amino Acid Sequence↗
PubMed2026

Novel CDK-independent function of CDC25 phosphatases in mRNA translation.

Molecular and functional networks driving coordination between cell cycle and mRNA translation remain to be explored. Here, we use mass spectrometry-based proteomics to comprehensively investigate the interactome and phosphoproteome of the cell cycle regulator CDC25A. We identify actors of mRNA regulation, such as RNA-binding proteins and translation factors, as interacting partners of CDC25A. CDC25A overexpression increases global translation, whereas catalytic inactivation or pharmacological inhibition decreases protein synthesis. A Cyclin-Dependent Kinase (CDK) interaction-deficient mutant of CDC25A also enhances translation, indicating a CDK-independent role. Our results further reveal an interplay between CDC25A and CDC25B whereby downregulation of CDC25A leads to compensatory overexpression of CDC25B. The roles of CDC25A and CDC25B in mRNA translation are independent of their roles in the cell cycle, with CDC25A possibly regulating translation elongation and CDC25B rather involved in initiation. In acute myeloid leukemia cells, CDC25A depletion also inhibits translation, suggesting its potential relevance as a therapeutic target. We propose that CDC25 phosphatases might be signaling platforms coordinating cell cycle progression with protein synthesis.

cdc25 Phosphatases↗
PubMed1999

Protein interactions at the tight junction. Actin has multiple binding partners, and ZO-1 forms independent complexes with ZO-2 and ZO-3.

Defining how the molecular constituents of the tight junction interact is a prerequisite to understanding tight junction physiology. We utilized in vitro binding assays with purified recombinant proteins and immunoprecipitation analyses to define interactions between ZO-1, ZO-2, ZO-3, occludin, and the actin cytoskeleton. Actin cosedimentation studies showed that ZO-2, ZO-3, and occludin all interact directly with F-actin in vitro, indicating that actin is engaged in multiple interactions at the tight junction. Low speed sedimentation analyses demonstrated that neither ZO-2, ZO-3, nor occludin act as F-actin cross-linking proteins, and further evidence indicates that these proteins do not bind to actin filament ends. The binding interactions of ZO-2, ZO-3, and occludin were corroborated in vivo by immunofluorescence colocalization experiments which showed that all three proteins colocalized with actin aggregates at cell borders in cytochalasin D-treated Madin-Darby canine kidney cells. Exploration of other tight junction protein interactions demonstrated that ZO-2 binds directly to both ZO-1 and occludin. Contrary to previous beliefs, our immunoprecipitation results indicate that ZO-1, ZO-2, and ZO-3 exist in situ primarily as independent ZO-1.ZO-2 and ZO-1.ZO-3 complexes rather than a trimeric ZO-1.ZO-2.ZO-3 grouping. These studies elucidate direct binding interactions among tight junction-associated proteins, giving insight into their organization as a multimolecular structure.

Actins↗
PubMed2006

The role of residue stability in transient protein-protein interactions involved in enzymatic phosphate hydrolysis. A computational study.

Finding why protein-protein interactions (PPIs) are so specific can provide a valuable tool in a variety of fields. Statistical surveys of so-called transient complexes (like those relevant for signal transduction mechanisms) have shown a tendency of polar residues to participate in the interaction region. Following this scheme, residues in the unbound partners have to compete between interacting with water or interacting with other residues of the protein. On the other hand, several works have shown that the notion of active site electrostatic preorganization can be used to interpret the high efficiency in enzyme reactions. This preorganization can be related to the instability of the residues important for catalysis. In some enzymes, in addition, conformational changes upon binding to other proteins lead to an increase in the activity of the enzymatic partner. In this article the linear response approximation version of the semimacroscopic protein dipoles Langevin dipoles (PDLD/S-LRA) model is used to evaluate the stability of several residues in two phosphate hydrolysis enzymes upon complexation with their activating partners. In particular, the residues relevant for PPI and for phosphate hydrolysis in the CDK2/Cyclin A and Ras/GAP complexes are analyzed. We find that the evaluation of the stability of residues in these systems can be used to identify not only active site regions but it can also be used as a guide to locate "hot spots" for PPIs. We also show that conformational changes play a major role in positioning interfacing residues in a proper "energetic" orientation, ready to interact with the residues in the partner protein surface. Thus, we extend the preorganization theory to PPIs, extrapolating the results we obtained from the above-mentioned complexes to a more general case. We conclude that the correlation between stability of a residue in the surface and the likelihood that it participates in the interaction can be a general fact for transient PPIs.

Adenosine Triphosphate↗
PubMed2004

Cellulose membrane supported peptide arrays for deciphering protein-protein interaction sites: the case of PIN, a protein with multiple natural partners.

Cellulose membrane supported peptide arrays, prepared according to the Spot method, allow the rapid identification and characterization of protein-protein interaction sites. Here, the method was used to screen reactive peptides from different proteins that bind to a single molecule, the PIN protein. PIN possesses two binding grooves, that have been shown to interact with several targets, including neuronal NO synthase, dynein intermediate chain, myosin V, the proapoptotic protein Bim, the scaffolding proteins DAP1alpha and gephyrin, and the transcription factor NRF-1. Arrays of peptides representing sequences of these targets were probed for reactivity with GST-tagged PIN, enabling the precise identification of binding motifs. Binding motifs were then minimized to seven or eight amino acid long peptides: YSKETQT for dynein IC, CDKSTQT for Bim, KDTGIQVD for nNOS, QSVGVQV for DAP1alpha and EDKNTMTD for myosin V. Alascan and substitution analysis provided proof that the Gln residue is critical for the interaction and cannot be easily replaced. Positions -1 and +1, just flanking the pivotal Gln, are also important; they consist of hydrophobic residues (Thr, Val) that could only be replaced by hydrophobic or aromatic amino acids. Position -4 is also critical for binding, with its Asp or Ser being replaceable to some extent. Alignment of sequences of proteins known to bind PIN shows that the most frequent amino acids in the motif are DKGTQT, consistent with the Spot results. We postulate that the degenerate character of binding to PIN is based on the propensity of several sequences to adopt a beta-strand conformation that allows the Gln residue to position itself in the PIN channel and on the conformational breathing of the PIN binding groove.

Amino Acid Sequence↗
PubMed2004

The adaptor protein Grb14 regulates the localization of 3-phosphoinositide-dependent kinase-1.

The metabolic actions of insulin are transduced through the phosphatidylinositol 3-kinase pathway. A critical component of this pathway is 3-phosphoinositide-dependent kinase-1 (PDK-1), a PH domain-containing enzyme that catalyzes the activating phosphorylation for many AGC kinases, including Akt and protein kinase C isozymes. We used a directed proteomics-based approach to identify the adaptor protein Grb14, which binds the insulin receptor through an SH2 domain, as a novel PDK-1 binding partner. Interaction of these two proteins is constitutive and mediated by a PDK-1 binding motif on Grb14. Disruption of this motif by point mutation or deletion of the Grb14 SH2 domain prevents the insulin-triggered membrane translocation of PDK-1. The interaction of PDK-1 with Grb14 facilitates Akt function: disruption of the interaction by overexpression of a construct of Grb14 mutated in the PDK-1 binding motif significantly decreases insulin-dependent activation of Akt. Thus, Grb14 serves as an adaptor protein to recruit PDK-1 to activated insulin receptor, thus promoting Akt phosphorylation and transduction of the insulin signal.

3-Phosphoinositide-Dependent Protein Kinases↗
PubMed2005

Major vault protein, in concert with constitutively photomorphogenic 1, negatively regulates c-Jun-mediated activator protein 1 transcription in mammalian cells.

Constitutively photomorphogenic 1 (COP1), a RING finger ubiquitin ligase with substrates including c-Jun and p53, was recently found to be overexpressed in a number of breast and ovarian tumor samples. In addition to its E3 activity, COP1 was also shown to be able to inhibit activator protein 1 (AP-1) transcription. Through an affinity purification method, we have identified major vault protein (MVP) as a novel interacting partner for COP1 in mammalian cells. MVP, also known as lung resistance protein, is the main component of a ribonucleoprotein organelle called vault, and has been implicated in multiple drug resistance in many cancer cell lines and primary tumor samples. The interaction between COP1 and MVP is detectable at the endogenous level and occurs mostly in the cytoplasm. Similar to COP1, MVP inhibits c-Jun accumulation and AP-1 transcription activity. MVP knockout or knockdown cells contain elevated amount of c-Jun and increased AP-1 transcription activity. UV irradiation enhances MVP tyrosine phosphorylation, causes dissociation of COP1 from MVP, and alleviates the inhibitory activity of MVP on AP-1 transcription. Taken together, we propose that MVP, most likely through its interaction with COP1, suppresses c-Jun-mediated AP-1 transcription under unstressed conditions, thereby preventing cells from undergoing stress response.

Amino Acid Sequence↗
PubMed2005

The histidine triad protein Hint1 interacts with Pontin and Reptin and inhibits TCF-beta-catenin-mediated transcription.

Pontin and Reptin previously were identified as nuclear beta-catenin interaction partners that antagonistically modulate beta-catenin transcriptional activity. In this study, Hint1/PKCI, a member of the evolutionary conserved family of histidine triad proteins, was characterised as a new interaction partner of Pontin and Reptin. Pull-down assays and co-immunoprecipitation experiments show that Hint1/PKCI directly binds to Pontin and Reptin. The Hint1/PKCI-binding site was mapped to amino acids 214-295 and 218-289 in Pontin and Reptin, respectively. Conversely, Pontin and Reptin bind to the N-terminus of Hint1/PKCI. Moreover, by its interaction with Pontin and Reptin, Hint1/PKCI is associated with the LEF-1/TCF-beta-catenin transcription complex. In this context, Hint1/PKCI acts as a negative regulator of TCF-beta-catenin transcriptional activity in Wnt-transfected cells and in SW480 colon carcinoma cells as shown in reporter gene assays. Consistent with these observations, Hint1/PKCI represses expression of the endogenous target genes cyclin D1 and axin2 whereas knockdown of Hint1/PKCI by RNA interference increases their expression. Disruption of the Pontin/Reptin complex appears to mediate this modulatory effect of Hint1/PKCI on TCF-beta-catenin-mediated transcription. These data now provide a molecular mechanism to explain the tumor suppressor function of Hint1/PKCI recently suggested from the analysis of Hint1/PKCI knockout mice.

Amino Acid Sequence↗
PubMed2006

Identification of a PDZ protein, PIST, as a binding partner for Rho effector Rhotekin: biochemical and cell-biological characterization of Rhotekin-PIST interaction.

Among various effector proteins for the small GTPase Rho, the function(s) of Rhotekin is (are) almost unknown. We have identified PIST [PDZ (PSD-95, Discs-large and ZO-1) domain protein interacting specifically with TC10 (a Rho-family small GTPase)] as a binding partner for Rhotekin, using yeast two-hybrid screening. Rhotekin was found to associate with PIST in vitro and in both polarized and non-polarized MDCK (Madin-Darby canine kidney) cells. The C-terminal SPV (Ser-Pro-Val) motif of Rhotekin exhibited binding to the PDZ domain of PIST. The binding was markedly inhibited by an activated version of Rho and partially by that of Rac or Cdc42 in COS7 cells. In contrast, TC10 had no effects on the binding. Immunofluorescence analyses revealed the co-localization of PIST and Rhotekin at the Golgi apparatus in non-polarized fibroblast-like MDCK cells and AJs (adherens junctions) in the fully polarized cells. PIST and Rhotekin are recruited from the cytosol to AJs as the cell becomes polarized. Expression of constitutively active Rho or prevention of Rhotekin-PIST interaction induced diffuse cytoplasmic distribution of Rhotekin in polarized MDCK cells. These results suggest that there is (1) Rho-dependent regulation of Rhotekin-PIST interaction, (2) involvement of PIST in the recruitment of Rhotekin to AJs and (3) a possible role(s) for these two proteins in cell-polarity development and/or maintenance.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Molecular complexity and dynamics of cell-matrix adhesions.

Currently >50 proteins have been reported to be associated with focal contacts and related ECM adhesions. Most of these contain multiple domains through which they can interact with different molecular partners, potentially forming a dense and heterogeneous protein network at the cytoplasmic faces of the adhesion site. The molecular and structural diversity of this 'submembrane plaque' is regulated by a wide variety of mechanisms, including competition between different partner proteins for the same binding sites, interactions triggered or suppressed by tyrosine phosphorylation, and conformational changes in component proteins, which can affect their reactivity. Indeed, integrin-mediated adhesions can undergo dynamic changes in structure and molecular properties from dot-like focal complexes to stress-fiber-associated focal contacts, which can further 'mature' to form fibronectin-bound fibrillar adhesions. These changes are driven by mechanical force generated by the actin- and myosin-containing contractile machinery of the cells, or by external forces applied to the cells, and regulated by matrix rigidity.

Cell Adhesion↗
PubMed1997

A conditionally expressed third partner stabilizes or prevents the formation of a transcriptional activator in a three-hybrid system.

We describe a three-hybrid system that involves three polypeptides that allow or prevent the formation of the transcriptional activator. Beside the two-hybrid fusion proteins, the third partner is under the control of the Met25 promoter, which is positively regulated in medium lacking methionine. We document a situation where such a third partner promotes interaction between two proteins, one fused to a DNA-binding domain and the other fused to an activator domain. This is demonstrated for cdk7-MAT1 interaction stabilized by the presence of cyclin H; these three polypeptides are found either free or associated with the transcription/DNA repair factor TFIIH. We also document the capacity of our system to conditionally inhibit the interaction between two polypeptides that otherwise elicit a positive two-hybrid response. This is demonstrated for Ras-Raf interaction precluded by an excess of Raf. The presence of a methionine-regulated promoter provides an "on" or "off" switch for the formation of the transcriptional activator, thus also providing an excellent control to evaluate the activation or inhibition properties of the third partner.

Artifacts↗